Nitrogen fixation in pine: Direct confirmation and location of the diazotrophic population

氮通常是自然生态系统中最受限制的植物养分，对能够通过生物固氮（BNF）提供自身供应的物种具有明显的优势。植物相关氮素可以满足某些非根瘤草本植物的部分植物氮需求，但已知的唯一可以从BNF中获得大部分植物氮（高达80%）的非根瘤植物包括几个甘蔗品种。甘蔗BNF的致病因子最初被认为是重氮养糖醋杆菌的内生菌株，但最近的研究表明，重氮养的主要来源可能涉及生活在植物组织上或内部的细菌联合体，可能包括未培养的菌株。根据甘蔗和其他草本植物中负责BNF的微生物是可培养内生菌的报道，我在具有重要商业价值的裸子植物——黑松中寻找了类似的微生物，并从黑松幼苗和树组织的表面灭菌提取物中分离出了几种具有显著乙炔还原活性的可培养多粘类芽孢杆菌菌株。其中一种菌株P2b-2R处理过的松树幼苗，从BNF中获得高达79%的叶面氮，积累的生物量显著高于对照。到目前为止，我的研究结果表明：(1)松树幼苗可以从P2b-2R的固氮中获得几乎所有的叶面氮；(2)如果幼苗生长足够长的时间，P2b-2R的BNF可以促进幼苗生长；(3)P2b-2R的内生习性有利于BNF。然而，目前所进行的实验是探索性的，没有直接测量N2固定，也没有排除其他可能促进幼苗生长的原因。我建议使用15N组织结合试验来证实我们的间接证据，即P2b-2R在松树中产生BNF，并使用野生型的P2b-2R nif衍生物来确定这种微生物对松树幼苗生长的促进是否来自BNF。此外，由于P2b-2R在内外根、茎和针组织中定植，我建议确定这些植物相关的N2固定群体中哪一个对松树相关的BNF贡献最大。

Nitrogen is often the most limiting plant nutrient in natural ecosytems rendering a distinct advantage to species able to provide their own supply through biological nitrogen fixation (BNF). Plant-associated BNF can satisfy part of the plant N requirement in certain non-nodulating herbaceous species but the only known non-nodulating plants that can obtain most plant N from BNF (up to 80%) comprise a few cultivars of sugarcane.The causative agent of BNF in sugarcane was initially thought to be an endophytic strain of Gluconoacetobacter diazotrophicus, but more recent studies indicate that the primary source of diazotrophy may involve a consortium of bacteria, possibly including uncultured strains, that live on or inside plant tissues. Based on reports than the microorganism responsible for BNF in sugarcane and other herbaceous plant species were culturable endophytes, I searched for similar microorganisms in the commercially important gymnosperm, lodgepole pine and isolated several culturable Paenibacillus polymyxa strains that possessed significant acetylene reduction activity from extracts of surface-sterilized lodgepole pine seedling and tree tissues. Pine seedlngs treated with one of these strains, P2b-2R, derived up to 79% of foliar N from BNF and accumulated significantly more biomass than controls. My results so far suggest that (i) pine seedlings can derive almost all foliar N from nitrogen fixation by P2b-2R, (ii) BNF by P2b-2R causes seedling growth promotion, if seedlings are grown for a sufficiently long time and (iii) P2b-2R's endophytic habit facilitates BNF. However, the experiments conducted so far were exploratory in nature and did not measure N2 fixation directly or rule out other possible causes of seedling growth promotion. I propose to confirm our indirect evidence of BNF by P2b-2R in pine using a 15N tissue incorporation assay and to determine if pine seedling growth promotion by this microorganism results from BNF using a P2b-2R nif- derivative of the wild type. In addition, because P2b-2R colonizes internal and external root, stem and needle tissues, I propose to determine which of these plant-associated N2 fixing populations contributes most to pine-associated BNF.